Pluripotent stem cells including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPS) are unique in that they can self-renew and/or differentiate into cells from every germ layer. Development of differentiated cells from either hESC or iPS could provide an unlimited supply of cells for use in patient therapy. However, surprisingly little is known about how to control growth and differentiation of either of these pluripotent cell types. In particular, the survival rate of hESCs after dissociation is less than 1%. This limits the ability to perform critical experiments including gene targeting, isolation of homogeneous populations of cells in feeder free conditions, and development of large scale differentiation protocols, each of which may be required prior to providing patient tailored therapy. In order to develop a greater understanding of how to improve survival of hESCs, we have added individual growth factors to growing cultures of cells to examine survival. However, this is not cost-effective, quantitative or comprehensive. Therefore in this proposal we will develop a high content screening (HCS) system to assay the potential of small molecules to improve survival. We will develop an OCT4-EGFP reporter line as OCT4 marks pluripotent stem cells and gets rapidly down regulated upon differentiation, as well as development of feeder free HCS assays to follow changes in stem cell numbers in a defined culture system. The second half of this proposal will be dedicated to validating potential targets found in the HCS screens using both molecular and cellular based assays to follow the effects of small molecule treatment on pluripotent stem cell survival. Development of HCS for either hESC or iPS is novel and could provide an excellent system in future applications for monitoring multiple cell lineages, genetic backgrounds and stem cell lines from patient-specifc disease models. PUBLIC HEALTH RELEVANCE: Understanding how pluripotent stem cells make decisions to survive or self-renew versus differentiate is not well understood. Prior to using differentiated cells obtained from stem cells we will need to develop better assays to understand how to maintain (or eliminate) these cells in culture. One mechanism of controlling this decision is to regulate how stem cells survive in culture. Therefore in this proposal we will develop a high content screening (HCS) assay, which will allow for examination of multiple parameters including stem cell survival, cell morphology and size as well as loss of pluripotency. The assays will also be developed in defined culture systems;therefore the effects of small molecules on stem cell survival will be direct to the stem cells in culture. The ability to control growth of pluripotent stem cells is critical prior to development of cell therapy for patients and the development of HCS will provide a critical tool for evaluating multiple stem cell lines in a uniform system.